Cyclisation process for the preparation of c-2 beta-lactam compounds

ABSTRACT

A process for the preparation of a substituted C-2 β-lactam comprises incubating a 2-substituted 3-aminocarboxylic acid with a β-lactam synthetase under conditions such that the 2-substituted 3-aminocarboxylic acid is cyclised to produce a substituted C-2 β-lactam. The process can be used to produce an antibiotic or β-lactamase inhibitor.

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of asubstituted C-2 β-lactam which comprises incubating a 2-substituted3-aminocarboxylic acid with a β-lactam synthetase or modified β-lactamsynthetase either in vitro or within cells. This process may be used inthe synthesis of β-lactam antibiotics.

BACKGROUND OF THE INVENTION

The β-lactam antibiotics, which include penicillins, cephalosporins andcarbapenems, contain a β-lactam ring as part of their chemicalstructure. This ring is vital to their antibiotic activity and iscritical in preventing peptides from attaching to side chains duringcell wall formation.

β-Lactam compounds are susceptible to degradation by β-lactamase enzymeswhich are produced by several clinically important microorganisms.β-lactamase inhibitors, such as clavulanic acid (1), have been usedsuccessfully in combination with β-lactam antibiotics in the treatmentof infections caused by β-lactamase producing microorgansims.

A β-lactam synthetase is believed to act in the biosynthetic pathway toclavulanic acid, in the cyclisation of N²-(2-carboxyethyl)-(L)-arginine(CEA) (2) to deoxyguanidinoproclavaminic acid (DGPC) (3).

However, whilst clavulanic acid and many other β-lactam antibiotics andβ-lactamase inhibitors based on the cephalosporins and penicillins maybe produced from fermented materials already containing β-lactam nuclei,other sub-families of β-lactam antibiotics, such as the β-lactamantibiotics, such as the carbapenems (e.g. (5)), are currently producedby total synthesis. For example, a key intermediate in the preparationof a number of β-lactam antibiotics, such as carbapenems, trinems andoxapenems, is 3-(1-hydroxyethyl)-4-(acetyloxy)-2-azetidinone (4), whichis produced by synthesis.

The production costs of antibiotics and β-lactamase inhibitors that mustpresently be prepared by total synthesis limits their use and precludesthe development of new compounds.

SUMMARY OF THE INVENTION

The present invention provides a process for the preparation of asubstituted C-2 β-lactam comprising incubating a 2-substituted3-aminocarboxylic acid with a β-lactam synthetase or modified β-lactamsynthetase under conditions such that the 2-substituted3-aminocarboxylic acid is cyclised to produce a substitutedC-2-β-lactam.

In a preferred embodiment the process further comprises synthesising acarbapenem, trinem or oxapenem antibiotic from the substituted C-2β-lactam so produced.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a process for the preparation of asubstituted C-2 β-lactam which uses a β-lactam synthetase enzyme.

A substituted C-2 β-lactam is formed by cyclisation of a 2-substituted3-aminocarboxylic acid by a β-lactam synthetase or a modified β-lactamsynthetase. Preferably, the 2-substituted 3-aminocarboxylic acidcomprises substituents which promote ring closure in the process of theinvention. The 2-substituted 3-aminocarboxylic acid is preferably amolecule of the general formula (I):

wherein R¹ is selected from C₁ to C₄ alkyl, C₁ to C₄ alkoxy, C₁ to C₄alkylthio, C₁ to C₄ hydroxyalkyl, amino, amido, amidino, guanidino,benzyl, phenyl, R¹³CONH, C₆H₅CH₂CONH and C₆H₅OCH₂CONH. In a preferredembodiment, R¹ is selected from methyl, ethyl, hydroxyethyl (of R or Sstereochemistry) and R¹³CONH; wherein R¹³ is selected from hydrogen,alkyl such as C₁ to C₁₀ alkyl, aryl, heteroaryl, aryl alkyl, aryl-C₂ toC₄ alkenyl or aryl-C₁ to C₄ alkyl such as an aryl methylene such asPhCH₂ or PhOCH₂; wherein aryl or heteroaryl are mono-ring, or have twofused rings one of which may be saturated, and which aryl and heteroarylgroups may be substituted by one or more C, to C₄ alkyl, halo, NR¹OR¹¹,SO₂R¹⁰R¹¹, CONR¹⁰R¹¹, C₁ to C₆ alkyl ester, CN, CH₂OH, O—C₁ to C₆ alkyl,CF₃ or nitro groups; wherein R¹⁰ and R¹¹, which may be the same ordifferent, are hydrogen or C₁ to C₄ alkyl; wherein R³ may be selectedfrom H, CH₃, CH₂CH₃, CO₂H, CONH₂, OAc, CO₂R¹⁴, CH₂OH and

wherein R¹⁴ is selected from hydrogen, alkyl, preferably C₁ to C₁₀alkyl, such as CH₃ or C₂H₅, and CH₂R¹⁵ wherein R¹⁵ is an aromatic groupsuch as phenyl, 4-nitrophenyl or methoxyphenyl or an aryl, heteroaryl,aryl alkyl, aryl-C₂ to C₄ alkenyl or aryl-C₁ to C₄ alkyl; wherein arylor heteroaryl are mono-ring, or have two fused rings one of which may besaturated, and which aryl and heteroaryl groups may be substituted byone or more C₁ to C₄ alkyl, halo, NR¹⁰R¹¹, SO₂R¹⁰R¹¹, CONR¹⁰R¹¹, C₁ toC₆ alkyl ester, CN, CH₂OH, O—C₁ to C₆ alkyl, CF₃ or nitro groups;wherein R¹⁰ and R¹¹, which may be the same or different, are hydrogen orC₁ to C₄ alkyl; and wherein R² is a readily functionalisable orcleavable group. Preferably, R² is selected from hydrogen and analiphatic or aromatic substituent, for example CH₂CO₂H, SO₂R⁷, CO₂R⁷,CONHR⁷, COR⁷, C═CR⁸R⁹ wherein R⁷ is C₁ to C₆ alkyl; CH₂(CF₂)₀₋₄CF₃; arylor heteroaryl, which aryl or heteroaryl are mono-ring, or have two fusedrings one of which may be saturated, and which aryl and heteroarylgroups may be substituted by one or more C, to C₄ alkyl, halo, NR¹⁰R¹¹,SO₂R¹⁰R¹¹, CONR¹⁰R¹¹, C₁ to C₆ alkyl ester, CN, CH₂OH, O—C₁ to C₆ alkyl,CF₃ or nitro groups; aryl-C₁ to C₄ alkyl; aryl-C₁ to C₄-alkyl-NH; oraryl-C₂ to C₄ alkenyl; or such groups wherein aryl is substituted by oneor more C₁ to C₄ alkyl or halo groups R⁸ and R⁹, which may be the sameor different, are COR¹²; R¹⁰ and R¹¹, which may be the same ordifferent, are hydrogen or C₁ to C₄ alkyl; and R¹² is hydrogen or analiphatic, aromatic or heteroaromatic group. Preferably, R² is of thegeneral formula —CR⁶—CO₂H, wherein R⁶ is hydrogen or an aliphatic group.Preferably R⁶ is of the general formula:

wherein R⁴ is selected from hydrogen and OH and R⁵ is selected from NH₂

Accordingly, in a preferred aspect, the 2-substituted 3-aminocarboxylicacid is of the formula:

wherein R¹ is as defined above.

The present invention provides a process by which a β-lactam synthetaseor modified β-lactam synthetase enzyme catalyses the cyclisation of a2-substituted 3-aminocarboxylic acid, for example a molecule of formula(I), to a substituted C-2 β-lactam, for example a molecule of formula(II):

In a preferred aspect of the invention, the substituted C-2 β-lactamformed by cyclisation of the 2-substituted 3-aminocarboxylic acid issuitable as an intermediate in the production of a β-lactam antibioticor β-lactamase inhibitor. For example, the substituted C-2 β-lactam maybe a 2-azetidinone such as3-(1-hydroxyethyl)-4-(acetyloxy)-2-azetidinone, or an intermediatesuitable for use in the formation of this compound.

The 2-substituted 3-aminocarboxylic acid may be obtained from anysuitable source, for example, by direct synthesis or by action ofanother enzyme either in vitro or in cells.

The present invention encompasses the use of 2-substituted3-aminocarboxylic acid or salts thereof in the processes describedherein. For example, the invention encompasses the salts of thosecompounds of formula (I) that have salt forming groups, such as anacidic or basic group. In the case of compounds comprising a basic aminogroup, the salts are formed with suitable inorganic or organic acids.Suitable inorganic acids are, for example, hydrochloric or sulphuricacid. Suitable organic acids include mono- di- and tricarboxylic acidssuch as acetic, trifluoroacetic, tartaric and citric acid, or sulphonicacids, for example methanesulphonic, trifluoromethanesulphonicorp-toluenesulphonic acid.

The present invention includes all possible isomers and mixturesthereof, including diastereomeric mixtures and racemic mixtures,resulting from the possible combinations of (R) and (S) stereochemistry.

For use in a process of the present invention, the 2-substituted3-aminocarboxylic acid or a salt thereof is suitably dissolved, forexample, in buffer before mixing with the enzyme. The concentration ofprecursor solution will depend upon the solubility of the precursor;usually the concentration of the precursor solution is in the range offrom 5% w/v to 0.001% w/v.

A β-lactam synthetase for use in connection with the present inventionis one has suitable β-lactam synthetase activity, that is, the abilityto catalyse cyclisation of an 3-aminocarboxylic acid, in particular an2-substituted 3-aminocarboxylic acid, to produce a β-lactam.

A β-lactam synthetase for use in the present invention may be anaturally occurring β-lactam synthetase, such as a β-lactam synthetaseenzyme purified from a natural source, for example from Streptomycesclavuligerus. Streptomyces antibioticus, or other clavam or carbapenemproducing Streptomyces species or from other microorganisms such asErwinia carotovora or Serratia marcesceus.

Variations in the sequence of β-lactam synthetase may be present inβ-lactam synthetase obtained from other microorganisms. A β-lactamsynthetase for use in the present invention may therefore be a naturallyoccurring variant which is expressed by Streptomyces clavuligerus oranother microorganism. Variant β-lactam synthetases include polypeptideswhich vary from the β-lactam synthetase of Streptomyces clavuligerus butare not necessarily naturally occurring β-lactam synthetases. Over theentire length of the amino acid sequence of the β-lactam synthetase ofStreptomyces clavuligerus, a variant will preferably be at least 80%homologous to that sequence based on amino acid identity. Morepreferably, the polypeptide is at least 85% or 90% and more preferablyat least 95%, 97% or 99% homologous to that amino acid sequence over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95%, amino acid identity over a stretch of 40 or more, forexample 60, 100 or 120 or more, contiguous amino acids (“hardhomology”).

Amino acid substitutions may be made to the β-lactam synthetase sequenceof Streptomyces clavuligerus, for example from 1, 2 or 3 to 10, 20 or 30substitutions. Conservative substitutions may be made, for example,according to the following table. Amino acids in the same block in thesecond column and preferably in the same line in the third column may besubstituted for each other: ALIPHATIC Non-polar G A P I L V Polar -uncharged C S T M N Q Polar - charged D E K R AROMATIC H F W Y

One or more amino acid residues of the β-lactam synthetase amino acidsequence of Streptomyces clavuligerus may alternatively or additionallybe deleted. From 1, 2 or 3 to 10, 20 or 30 residues may be deleted, ormore. Enzymes for use in the processes of the invention also includefragments of the above-mentioned sequences retain β-lactam synthetaseactivity. Fragments may be at least from 10, 12, 15 or 20 to 60, 100 or200 amino acids in length.

A β-lactam synthetase enzyme may be obtained from an organism such asStreptomyces clavuligerus by culturing the microorganism, harvesting andlysing the mycelium, and isolating the β-lactam synthetase enzyme.β-lactam synthetase from other Streptomyces clavuligerus strains orother bacterial species can be isolated following standard cloningtechniques, for example, using the polynucleotide sequence of β-lactamsynthetase from Streptomyces clavuligerus or a fragment thereof as aprobe.

The β-lactam synthetase may be used in vitro or in cells. A cell-freeenzyme extract may be produced, for example by sonication or otherdisruption of the microorganisms, optionally thereafter removing celldebris, leaving the β-lactam synthetase enzyme in solution. Thissolution may then be fractionated to isolate the β-lactam synthetaseenzyme. The enzyme may be isolated and used in purified form,partially-purified form, as obtained in an impure state, as a filtratefrom a disrupted cell preparation, as a crude cell homogenate, or in animmobilised form on a column.

For use in vitro, the enzyme may be in a substantially isolated form. Itwill be understood that the enzyme may be mixed with carriers ordiluents which will not interfere with the intended purpose of theenzyme and still be regarded as substantially isolated.

The enzyme may also be in a substantially purified form, in which caseit would generally comprise the enzyme in a preparation in which morethan 90%, e.g. 95%, 98% or 99%, by weight of the enzyme in thepreparation is an active β-lactam synthetase enzyme. Most suitably theenzyme is, for example, at least partially purified to remove otherenzymes which might catalyse the destruction of the precursor, theenzyme, or the β-lactam nucleus once formed.

The enzyme may be recovered and purified from the disrupted cellpreparation by well-known methods including ammonium sulphate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,size exclusion chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high-performance liquid chromatography or a variantoptimised for protein purification is employed for purification.Well-known techniques for refolding proteins may be employed toregenerate an active conformation when the polypeptide is denaturedduring isolation and/or purification.

The enzyme may be made synthetically or by recombinant means. The aminoacid sequence of the enzyme may be modified to include non-naturallyoccurring amino acids or to increase the stability of the enzyme. Wherethe enzyme is produced by synthetic means, such amino acids may beintroduced during production. The enzyme may also be modified followingeither synthetic or recombinant production. A modified β-lactamsynthetase suitable for use in a process of the present invention willretain the activity of a β-lactam synthetase to cyclise a3-aminocarboxylic acid, in particular the ability to cyclise a2-substituted 3-aminocarboxylic acid.

The β-lactam synthetase enzyme may be modified for example by deletionor mutation such that the β-lactam synthetase activity is modified orincreased. In particular, the activity of the enzyme for a substrateother than its natural substrate may be increased. For example, themodification may enhance the activity or specificity of the enzyme for asubstrate which is substituted at the C-2 position. Preferably themodified β-lactam synthetase demonstrates improved activity in thecyclisation of a 2-substituted 3-aminocarboxylic acid to a substitutedC-2 β-lactam compared to the corresponding enzyme which does notincorporate the selected modification. The β-lactam synthetase may bemodified through amino acid substitution within the active site of theenzyme.

A β-lactam synthetase enzyme may be modified, for example by theaddition of histidine residues to assist its identification orpurification or by addition of a signal sequence to promote itssecretion from a cell where the enzyme does not naturally contain such asequence.

The β-lactamase enzyme may be modified by, for example, amino acidsubstitution, deletion or extension. A modified β-lactam synthetaseenzyme may be a fragment of a naturally-occurring β-lactam synthetaseenzyme which retains the β-lactam synthetase activity. The β-lactamsynthetase enzyme may be extended at the N-terminus or C-terminus of theamino acid sequence of the enzyme. A carrier protein may be fused to theenzyme. A fusion protein incorporating an active β-lactam synthetase canthus be provided.

A number of side-chain modifications are known in the art and may bemade to the side-chains of the enzyme. Such modifications include, forexample, modifications of amino acids by reductive alkylation byreaction with an aldehyde followed by reduction with NaBH₄, amidinationwith methylacetimidate or acylation with acetic anhydride.

The amino acid sequence encoding the β-lactam synthetase enzyme may beintroduced into a cell by in situ expression of the polypeptide from arecombinant expression vector. The expression vector optionally carriesan inducible promoter to control the expression of the polypeptide.

An enzyme may be used in vitro, for example, bound to an immobilesubstrate such as an insoluble polymeric support. The enzyme may beimmobilised through the addition of a binding sequence such as a His-tagor maltose binding site or by using a general immobiliser. Theimmobilised enzyme can then be used in the processes of the invention.

A compound or enzyme for use in the present invention may carry arevealing label. The revealing label may be any suitable label whichallows the compound or enzyme to be detected. Suitable labels includeradiostopes such as ¹²⁵I, ³³P or ³⁵S, enzyme labels, antibodies orlinkers such as biotin. Such labels may be detected using techniquesknown per se.

A β-lactam synthetase or modified β-lactam synthetase in accordance withthe invention is useful in the cyclisation of 2-substituted3-aminocarboxylic acids. Such cyclisation may be carried out in vitro orin vivo. Such cyclisation may be used as part of the process for theproduction of a β-lactam antibiotic or β-lactamase inhibitor.

The process of the present invention is generally carried out in aqueousmedia, the reaction mixture suitably being maintained in the range offrom pH 4 to 9, more suitably, for example, from 6.5 to 9.0, preferablyabout pH 8.5. The pH is suitably controlled, for example, using buffers,such as, for example, 3-(N-morpholino)propanesulphonic acid ortris(hydroxymethyl)aminomethane buffer at pH 9. Alternatively the pH maybe controlled by the addition of a suitable acid or base. Thetemperature of the reaction should be that suitable for the enzymeemployed and is generally in the range of from 15° C. to 60° C.,preferably about 30-37° C. The reaction time depends on such factors asconcentrations of reactant and cofactors, temperature and pH.

After the reaction is complete, the enzyme may be separated from thereaction mixture and the substituted C-2 β-lactam or a salt thereof,isolated by conventional methods, such as by chromatography, extractioninto an organic solvent or by precipitation. The substituted C-2β-lactam may be isolated in a form where the carboxyl and/or the aminogroup present is protected and, if desired, the protecting group(s) maybe subsequently removed to generate the compound in a pure form.

Salts of the substituted C-2 β-lactam may be produced, for example, bytreating the unsalified compound with the appropriate acid or base. Thecompounds, and salts thereof, produced by the above processes, may berecovered by conventional methods.

Substituted C-2 β-lactam possessing two chiral centres may be separatedinto diastereoisomeric pairs of enantiomers, if so desired, by, forexample, fractional crystallisation from a suitable solvent, for examplemethanol or ethyl acetate or a mixture thereof. The pair of enantiomersor other pairs of enantiomers may be separated into individualstereoisomers by conventional means, for example by the use of anoptically active salt as a resolving agent or by stereoselective removalof a protecting group using a suitable enzyme, for example an esterasesuch as subtilisin. In mixtures of diastereoisomers of the compounds theratio of diastereoisomers may be changed by treatment with anon-nucleophilic base, for example, 1,5-diazabicyclo[4.3.0]non-5-ene.

Suitable optically active compounds which may be used as resolvingagents are described in ‘Topics in Stereochemistry’, Vol.6, WileyInterscience, 1971, Allinger, N. L. and Eliel, W. L., Eds.

Alternatively, any enantiomer of a substituted C-2 β-lactam may beobtained by stereospecific synthesis using optically pure substituted4-amino carboxylic acid of known configuration.

In an alternative aspect of the invention, host cells may be providedwhich are transformed with polynucleotide encoding a β-lactam synthetaseor modified β-lactam synthetase suitable for use in a process of thepresent invention. A polynucleotide encoding a β-lactam synthetase maybe introduced into a replicable vector, for example a vector which iscapable of providing for the expression of the coding sequence by thehost cell. The vector may be for example, a plasmid, virus or phagevector provided with an origin of replication, optionally a promoter forthe expression of the said polynucleotide and optionally a regulator ofthe promoter. The vectors may contain one or more selectable markergenes, for example a tetracycline resistance gene. Promoters and otherexpression regulation signals may be selected to be compatible with thehost cell for which the expression vector is designed. Multiple copiesof the same or different β-lactam synthetase gene in a single expressionvector; or more than one expression vector each including a β-lactamsynthetase gene which may be the same or different may be transformedinto the host cell. The vector may then be introduced into a compatiblehost cell and the cell cultivated under conditions which bring about theexpression of the polypeptide. Preferably, the host cells will be cellsof bacterial or fungal origin such as E. coli, a Streptomyces SPP orPeincillium chrysogenum, particularly, for the production in vitro ofthe β-lactam synthetase for use in the invention.

The invention may therefore be operated using such an intact host cellexpressing a β-lactam synthetase enzyme. The precursor 2-substituted3-amino carboxylic acid, or salt thereof, is provided and contacted withthe microorganism under conditions enabling conversion of the2-substituted 3-amino carboxylic acid to the substituted C-2 β-lactam.Alternatively, the enzyme may be expressed in a host cell which iscapable of producing a 2-substituted 3-aminocarboxylic acid such thatthe 2-substituted 3-aminocarboxylic acid is converted to a substitutedC-2 β-lactam.

The host cell may be in the form of a growing culture, resting culture,washed mycelium, immobilised cells, or protoplasts.

In a further embodiment a cell-free system, derived from the recombinantorganism, may be used to carry out the process of the invention. Thecell-free extract may be prepared, and the enzyme purified, ashereinbefore described.

The substituted C-2 β-lactam obtained by the processes of the presentinvention may be active as an antibiotic, for example a carbapenem,trinem or oxapenem antibiotic or as a β-lactamase inhibitor. In anotheraspect, the substituted C-2 β-lactam produced by the processes of theinvention is an intermediate in the synthesis pathway of an antibiotic,for example, a carbapenem, trinem or oxapenem antibiotic or of aβ-lactamase inhibitor. The present invention therefore also provides aprocess wherein an antibiotic or β-lactamase inhibitor is synthesisedfrom the C-2 β-lactam. Preferably, a carbapenem, trinem or oxapenemantibiotic is synthesised from the C-2 β-lactam. Synthesis may becarried out by routine methods known to the person skilled in the art.The following examples further illustrate the present invention:

EXAMPLES

2-Methyl CEA was prepared by substituting methyl acrylic acid foracrylic acid in the reported method for preparation of CEA (Baldwin, J.E.; Lloyd, M. D.; Whason, B.; Schofield, C. J.; Elson, S. W.; Baggaley,K. H.; Nicholson, N. H. J. Chem. Soc., Chem. Commun. 1993, 500-502).

Michael reaction of protected ornithine (7) with 2-methyl acrylic acid,followed by ring closure and deprotection gave epimeric monocyclicβ-lactam (8). Guanylation followed by hydrolysis gave the desiredanalogue (6) as a mixture of epimers.

The reagents for each of the steps shown above were as follows: (i)CH₂═CMeCO₂H, MeCN, 60° C., 60%; (ii) MeSO₂Cl, NaHCO₃(aq.), MeCN, 60° C.;(iii) 10% Pd/C/H₂, EtOH:H₂O (2:1); (iv)1-amidino-3,5-dimethylpyrazole-HNO₃, dimethylformamide-H₂O, pH 8-9, 45%plus 40% recovered (8); (v) 1M HCl 1 hr.

The bls gene has previously been expressed at relatively low levelsusing a pET24a(+) construct and purified by an involved two columnprotocol giving low overall yields (McNaughton, H. J.; Thirkettle, J.E.; Zhang, Z. H.; Schofield, C. J.; Jensen, S. E.; Barton, B.; Greaves,P. J. Chem. Soc., Chem. Commun. 1998, 2325-2326; Bachmann, B. O.;Townsend, C. A. Biochemistry 2000, 39, 11187-11193). To facilitatepurification a polyhistidine tagged form of BLS (β-lactam synthetase)was produced by cloning bis into the pET28a(+) vector (from Novagen).BLS produced using this vector was isolated to >95% purity (by SDS PAGEanalysis) using a single affinity purification step with a Nickel Hisbind™ column.

The bls gene in the vector pET28a(+) and a plasmid bearing thechaperonin GroELS¹³ were simultaneously transformed into E. coliBL21(DE3) for co-expression. Cells were grown in 2TY containing 30 μg/mlkanamycin at 37° C. When the absorbance at 600 nm reached ˜1.0, IPTG wasadded to a final concentration of 1.0 mM and the cells harvested 16hours later by centrifugation. Expression of BLS was observed as 15% ofthe total cell protein in the soluble fraction (by SDS-PAGE analysis).Cells were resuspended in binding buffer (20 mM Tris-HCl pH 7.9, 500 mMNaCl, 5 mM imidazole) and lysed by sonication. The lysate wascentrifuged at 40000 g, and the supernatant loaded onto apre-equilibrated 10 ml HisBind™ column. The column was washed with 100ml binding buffer followed by 60 ml wash buffer (binding buffer with 60mM imidazole). Protein was eluted with elute buffer (binding buffer with200 mM imidazole). The protein was desalted through a PD-10 column(Pharmacia) and concentrated to 10 mg/ml. BLS was aliquoted into 50 μlportions and stored at −80° C.

The 2-methylated substrate analogue (6) was initially assayed as asubstrate for BLS using a fluorescence detection pre-column derivatisedHPLC assay based on the work of Kai et al (Kai, M.; Miyazaki, T.;Yamaguchi, M.; Ohkura, Y. J. Chromatogr. 1983, 425-436). Assay mixturescontained: 150 mM Tris-HCl, pH 9, 5 mM ATP, 10 mM MgCl₂, 2 mM 2-methylCEA, ca. 74 μg enzyme.

Reactions were incubated at 37° C. for 15 minutes and then derivatised.Derivatisation conditions: To 50 μl assay mixture were added 25 μlbenzoin (40 mM) in 2-methoxyethanol, 25 μl sodium sulphite (0.2M)/β-mercaptoethanol (0.1 M), 50 μl KOH (2 M). The mixture was cooled onice for 2 minutes, heated to 100° C. for 5 minutes and then cooled onice for a further 2 minutes. 50 μl of Tris-HCl pH 9.2 was then added andthe sample centrifuged for 30 seconds at 11000 g. 150 μl of the abovesample was injected on to a reverse phase Hypersil phenyl column (250mm×4.6 mm) and the elution of compounds compared to that of standards. Alinear gradient of 15% (v/v) Tris-HCl (0.5 M) pH 8.5, 50-80% (v/v) MeOHover 16 minutes remaining at 80% (v/v) MeOH for 6 minutes, reverting to50% (v/v) MeOH over 1 minute and re-equilibrating under these conditionsfor a further 15 minutes was run at 0.7 ml/min. Fluorescence wasmeasured at 425 nm against 325 nm excitation.

Under the standard assay conditions ca. 50% of the 2-methyl CEA (6) wasconverted to 2-methyl DGPC (9), which was observed as a single HPLC peakcoincident with that produced when using authentic (9). The HPLC assayswere also carried out using wildtype BLS to ensure the polyhistidine tagwas not affecting the conformation of the protein such that it acceptedthe alternative substrate. A similar result was obtained.

Incomplete substrate conversion may have in part resulted from partialepimerisation at the α-centre derived from arginine during the ringclosure process in the synthesis, or from preferential processing of oneof the 2-methyl epimers of (6). To investigate this a larger scaleincubation was used to allow analysis by ¹H NMR (500 MHz). Peaks wereobserved at δ 4.2-4.5 consistent with conversion of ATP to AMP (FIG. 3).

The appearance of peaks corresponding to 2-methyl DGPC product (9) wasalso observed, including δ 4.05 (epimer 1, dd, J 10.0, 4.5 Hz, CHCO₂H),δ 4.0 (epimer 2, dd, J9.5, 5.5 Hz, CHCO₂H) and δ 1.5-2.0 (CH₂CH₂CH₂N &CH₂CH₂CH₂N)(FIG. 4).

The β-lactam product was isolated by reverse phase HPLC (C18 250×10 mm).Samples were eluted isocratically with a mobile phase of 10% (v/v) MeOHat a flow rate of 4 ml/minute. ESI MS analysis of the product wasconsistent with the production of 2-methyl DGPC (9) (m/z 243 [M−H]⁺). ¹HNMR (500 MHz), including 2D ¹H COSY analysis of the product wasconsistent with the formation of both 2 -methyl epimers of the product.There appeared to be a slight excess (<5%) of one epimer in the startingmaterial (6) (by ¹H NMR analysis), that was apparently also present inthe enzymic product (9), indicating that both epimers are approximatelyequally efficient substrates for BLS.

1. A process for the preparation of a substituted C-2 β-lactamcomprising incubating a 2-substituted 3-aminocarboxylic acid with aβ-lactam synthetase under conditions such that the 2-substituted3-aminocarboxylic acid is cyclised to produce a substituted C-2β-lactam.
 2. A process according to claim 1 wherein said 2-substituted3-aminocarboxylic acid is of the general formula

wherein R¹ is selected from C₁ to C₄ alkyl, C₁ to C₄ alkoxy, Ca to C₄alkylthio, C₁ to C₄ hydroxyalkyl, amino, amidino, guanidino, benzyl,phenyl, R¹³CONH, C₆H₅CH₂CONH and C₆H₅OCH₂CONH; wherein R¹³ is selectedfrom hydrogen, alkyl, aryl; heteroaryl, aryl alkyl, aryl-C₂ to C₄alkenyl or aryl-C₁ to C₄ alkyl; wherein aryl or heteroaryl aremono-ring, or have two fused rings one of which may be saturated, andwhich aryl and heteroaryl groups may be substituted by one or more C₁ toC₄ alkyl, halo, NR¹⁰R¹¹, SO₂R¹⁰ R¹¹, CONR¹⁰R¹¹, C₁ to C₆ alkyl ester,CN, CH₂OH, O—C₁ to C₆ alkyl CF or nitro groups: wherein R¹⁰ and R¹¹,which may be the same or different, are hydrogen or C₁ to C₄ alkyl;wherein R² is selected from hydrogen and an aliphatic or aromaticsubstituent; and wherein R³ is selected from hydrogen, CH₃, CH₂CH₃,CO₂H, CONH₂, OAc, CO₂R¹⁴, CH₂OH and

wherein R¹⁴ is selected from hydrogen, alkyl and CH₂R¹⁵ wherein R¹⁵ isan aromatic group.
 3. A process according to claim 2 wherein R¹ isselected from methyl and hydroxyethyl.
 4. A process according to claim 2wherein R² is of the general formula —CR⁶—CO₂H, wherein R⁶ is hydrogenor an aliphatic group.
 5. A process according to claim 4 wherein R⁶ isof the general formula

wherein R⁴ is selected from hydrogen or —OH and R⁵ is selected from


6. A process according to claim 1 wherein said β-lactam synthetase is anaturally occurring β-lactam synthetase.
 7. A process according to claim6 wherein said β-lactam synthetase is obtained from Streptomycesclavuligerus.
 8. A process according to claim 1 wherein said β-lactamsynthetase is a modified β-lactam-synthetase.
 9. A process according toclaim 8 wherein said modification increases the β-lactam synthetaseactivity for the 2-substituted 4-aminocarboxylic acid.
 10. A processaccording to claim 1 which is carried out in vitro.
 11. A processaccording to claim 1 wherein said β-lactam synthetase is provided by ahost cell which expresses the β-lactam synthetase.
 12. A processaccording to claim 1 wherein said substituted C-2 β-lactam is anantibiotic or β-lactamase inhibitor.
 13. A process according to claim 1further comprising synthesising an antibiotic or β-lactamase inhibitorfrom said substituted C-2 β-lactam.